Tissue factor (TF), a receptor of the coagulation factor VII expressed on the cell surface, plays an indispensable role in the activation of coagulation factors IX and X through the formation of a complex with the coagulation factor VII, and has been defined as a practical initiating factor of blood coagulation reactions.
TF is known to be expressed in fibroblasts, smooth muscle cells, etc. that constitute the blood vessel, and to play a hemostatic function by activating the coagulation system at the time of blood vessel injury.
DIC is a disease in which the activation of the coagulation system in a blood vessel leads to systemic multiple occurrence of blood clots, mainly in the microvasculature. It is not uncommon that the reduction of platelets and coagulation factors due to consumption leads to bleeding which is the opposite phenomenon to blood clotting. The multiple microthrombi can cause deficient microcirculation in the major organs, which, once developed, leads to irreversible functional deficiency and to bad prognosis of DIC, and in this sense DIC is considered an important disease.
The incidence of underlying diseases estimated from the 1990 and 1992 research reports by the Ministry of Health and Welfare Specified Diseases Blood Coagulation Disorders Survey and Study Group is: hematological malignancies, about 30%; solid tumors about, 20%; infections, about 15%; obstetric diseases, about 10%; hepatic diseases about, 6%; shocks, about 5%; and cardiovascular diseases, about 3%. The incidence of DIC is as high as about 15% in leukemia and about 6 to 7% in malignant lymphoma, and about 3% in solid tumors.
DIC develops accompanied by various diseases mentioned above, but the causative agent thereof is the same, which is TF. Thus, the onset mechanism of DIC is believed to be: abnormally high formation and/or expression of TF in cancer cells in acute leukemia, malignant lymphoma, and solid tumors; the enhanced formation and/or expression of TF in monocytes and/or endothelial cells in infections (in particular, sepsis caused by Gram-negative bacilli); TF influx into the blood from the necrotized liver tissue in fulminant hepatitis; TF expression on the lumina of the blood vessel in aortic aneurysm, cardiac aneurysm, and giant hemangioma; and also TF influx into the blood in obstetric diseases (amniotic fluid embolism and abruptio placentae) and surgeries, injuries, and burns.
The treatment of the original (underlying) diseases is of utmost concern, which, however, is not easy in practical terms.
As a current method of treating DIC, anticoagulant therapy and substitution therapy are in use. Heparin preparations (fractionated heparin, low molecular weight heparin) are mainly used for anti-coagulant therapy. Synthetic protease inhibitors (gabexate mesilate, nafamostat mesilate) and concentrated plasma (antithrombin III, activated protein C preparations) are also used. As a substitution therapy, there are platelet concentrates, fresh frozen plasmas (supply of fibrinogen), washed red blood cells, and the like.
However, the current therapeutic agents are not satisfactory in terms of efficacy and side effects, and in most cases complete withdrawal from DIC is impossible. Therefore, there is a need for the use of drugs having high therapeutic effects and low side effects.
On the other hand, as new attempts in DIC treatments there can be mentioned thrombomodulin preparations, hirudin, and anti-PAF agents, Tissue factor pathway inhibitor (TFPI). FXa-selective inhibitors are attracting attention as orally administrable anticoagulant and/or antithrombotic agents. Also as an agent that neutralizes the activity of TF, WO 88/07543 discloses mouse anti-human TF monoclonal antibody, and WO 96/40921 discloses humanized anti-human TF antibody.
Mouse anti-human TF monoclonal antibodies are expected to make a safe and effective therapeutic agent in that it does not exhibit a symptom of bleeding associated with main efficacy in DIC. However, mouse monoclonal antibodies are highly immunogenic (sometimes referred to as “antigenic”), and thus the medical therapeutic value of mouse antibodies in humans is limited. For example, the half life of mouse antibodies in humans is relatively short and therefore they cannot fully exhibit their anticipated effects. Furthermore, human anti-mouse antibody (HAMA) that develops in response to the mouse antibody administrated causes immunological reactions that are unfavorable and dangerous to patients. Thus, mouse monoclonal antibodies cannot be repeatedly administered to humans.
In order to solve these problems, methods have been developed that intend to reduce the immunogenicity of antibodies derived from non-humans, such as (monoclonal antibodies derived from) mice. One of them is a method of making chimeric antibody in which a variable region (v region) of the antibody is derived from mouse monoclonal antibody, and a constant region (C region) thereof is derived from a suitable human antibody.
Since the chimeric antibody obtained contains variable region of an original mouse antibody in the complete form, it is expected to bind to an antigen with the identical affinity as that of the original mouse antibody. Furthermore, in chimeric antibody the ratio of the amino acid sequences derived from non-humans is substantially reduced, and thereby it is expected to have a reduced immunogenicity compared to the original mouse antibody. However, it is still possible for an immunological response to the mouse variable region to arise (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86: 4220-4224, 1989).
A second method of reducing the immunogenicity of mouse antibody is, though much more complicated, expected to drastically reduce the potential immunogenicity of mouse antibody. In this method, the complementarity determining region (CDR) alone of a mouse antibody is grafted onto a human variable region to make a “reshaped” human variable region. As desired, however, some amino acid sequences of framework regions (FRs) supporting the CDRs may be grafted from the variable region of a mouse antibody onto the human variable region in order to obtain the closest possible approximation of the original mouse antibody structure. Then, the humanized reshaped human variable region is ligated to the human constant region. In the finally reshaped humanized antibody, portions derived from non-human amino acid sequences are only the CDRs and a small portion of the FRs. The CDRs comprise hypervariable amino acid sequences and they do not show species-specific sequences.
For humanized antibody, see also Riechmann, L. et al., Nature 332: 323-327, 1988; Verhoeye, M. et al., Scienece 239: 1534-1536, 1998; Kettleborough, C. A. et al., Protein Engng., 4: 773-783, 1991; Maeda, H., Human Antibodies and hybridoma, 2: 124-134, 1991; Gorman, S. D. et al, Proc. Natl. Acad. Sci. USA, 88: 4181-4185, 1991; Tempest, P R., Bio/Technology, 9: 226-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA 88: 2869-2873, 1991; Cater, P. et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148: 1149-1154, 1992; and Sato, K. et al., Cancer Res., 53: 851-856, 1993.
In the conventional humanization technology, part of the framework region (FR) includes an amino acid sequence that was grafted from the variable region of a mouse antibody to the human variable region. Thus, when it is administered as a therapeutic agent in humans, there is a risk that antibodies are formed against a site having an amino acid sequence not present in humans, though it is merely one to a few amino acids. In order to circumvent the risk, a third humanization technology was devised. Thus, the method involves, for four FRs (FR1-4) required to support the three dimensional structure of three CDRs, the substitution of the FR of a human antibody having a high homology with the FR of the mouse antibody present in the database using one FR as a unit. In this case, several FRs are selected from human antibodies present in the database, and are sequentially shuffled to prepare a humanized antibody having a high activity.
By so doing, it is possible to construct humanized antibodies in which all the FRs except the CDRs in the variable region have amino acid sequences derived from human antibody. Thus, the humanized antibody carrying the mouse CDR should no longer have immunogenicity more potent than a human antibody containing the human CDR.
Although humanized antibody is expected to be useful for the purpose of treatment, as mentioned above, there is no fixed process present which is universally applicable to any antibody in the method of producing humanized antibody, and thereby various contrivances are required to construct a humanized antibody that exhibits a sufficient binding activity and neutralizing activity to a specific antigen (see, for example, Sato, K. et al., Cancer Res., 53: 851-856, 1993).